Abstract
Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer–ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.
Highlights
Riboswitches are noncoding mRNA sequences usually found in the 5 untranslated regions of many genes involved in metabolite biosynthesis or transport, which they regulate by binding specific, related metabolites [1,2]
Given the lack of data regarding the intracellular concentration of Mg2+ ions in B. subtilis, we determine the Mg2+ concentration range in growth-phase B. subtilis, relevant to lysC riboswitch function in vivo, using inductivelycoupled plasma mass spectrometry (ICP-MS)
We found that the addition of only 75 M lysine ligand was enough to induce a shift of ∼65% of the lysC aptamer population into the high-FRET state (Figure 2D, bottom panel and Supplementary Figure S9a)
Summary
Riboswitches are noncoding mRNA sequences usually found in the 5 untranslated regions of many genes involved in metabolite biosynthesis or transport, which they regulate by binding specific, related metabolites [1,2]. Riboswitch architecture includes an aptamer domain that acts as the metabolite-sensing element and a downstream expression platform that interacts with the transcription or translation machinery [3,4,5]. A cascade of local and long-range conformational changes is initiated by ligand binding to the aptamer and transmitted to the expression platform [6], biasing its structure towards one of two competing conformers that control the activation or repression of the downstream gene (Figure 1A). Riboswitches have been shown to regulate gene expression through different mechanisms including transcription termination, translation inhibition and, in some eukaryotes, by exposing an alternative mRNA splicing site [1,5,7].
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